GSM7118579: Vehicle - J17B_TBI; Mus musculus; RNA-Seq
Lesional-perilesional brain tissue was removed (Bregma +0.64 mm to -2.86 mm) and immediately frozen on dry ice. Total RNA was isolated using RNeasy plus micro kit (Qiagen). Quantity of total RNA was assessed with Qubit 2.0 and quality was checked using a RNA 6000 Nano chip on Agilent's bioanalyzer. 200 ng of total RNA was send to Novogene (Cambridge, UK) for library preparation and sequencing. Barcoded mRNA seq libraries were prepared from 200 ng total RNA. For library preparation at Novogene, the mRNA present in the total RNA sample was isolated with magnetic beads of oligos d(T)25. This method is known as polyA-tailed mRNA enrichment. Subsequently, mRNA was randomly fragmented and cDNA synthesis proceeded through the use of random hexamers and the reverse transcriptase enzyme. Once the synthesis of the first chain was finished, the second chain was synthesized with the addition of an Illumina buffer. With this and together with the presence of dNTPs, RNase H and polymerase I from E.coli, the second chain was obtained by Nick translation. The resulting products went through purification, end-repair, A-tailing and adapter ligation. Fragments of the appropriate size were enriched by PCR, where indexed P5 and P7 primers were introduced, and final products were purified. The library was checked with Qubit 2.0 and real-time PCR for quantification and bioanalyzer Agilent 2100 for size distribution detection. Quantified libraries have been pooled and sequenced on Illumina Novaseq 6000 platform, according to effective library concentration and data amount. Sequencing strategy was paired-end 150 cycles (PE150).